The Ca2+-calcineurin-NFAT pathway is essential for the adaptive immune response, a point underscored by the clinical efficacy of the calcineurin inhibitors cyclosporin A (CsA) and FK506. In T cells, the transcription factor NFAT translocates from the cytoplasm into the nucleus in response to Ca2+ entry through `CRAC' channels, which are opened by the calcium sensors STIM1 and STIM2 in response to depletion of endoplasmic reticulum (ER) Ca2+ stores. During the previous two funding periods of this project, we used genome-wide RNAi screening in Drosophila and functional studies in human and mouse T cells to demonstrate that the long-sought CRAC channel is assembled from ORAI-family proteins; showed that a mutation in ORAI1 is responsible for a hereditary human immunodeficiency disease; and established by mutational analysis that ORAI1 is the pore subunit of the CRAC channel. We generated gene-disrupted mice with a complete deletion of ORAI1 or with conditional (`floxed') alleles of the ER Ca2+ sensors STIM1 or STIM2 and analyzed their immune phenotypes. We also performed a genome-wide RNAi screen in human cells (HeLa cells) that identified multiple proteins that modulate NFAT activation. Followup to this screen demonstrated that septins are essential modulators of the store-operated STIM-ORAI Ca2+ entry pathway in cells. Through biochemical studies of STIM1, ORAI1, and their protein-protein interactions, we showed that the cytoplasmic region of STIM1 by itself suffices to gate the ORAI1 channel; mapped the pore-lining transmembrane helices of the ORAI1 channel; and unraveled the mechanism through which dimerization of the ER-luminal domains of STIM1 triggers a conformational change in the cytoplasmic domains of STIM1, and thereby activates STIM to open the ORAI channel. In this proposal, we will build on these findings to extend our understanding of STIM-ORAI signalling and Ca2+ entry pathways in T cells. In Aim 1, we will investigate ORAI1 channel gating using purified STIM1, ORAI1, and protein modulators. For this, we are developing and utilizing state-of-art-techniques for monitoring protein- protein interactions and protein conformational changes. In Aim 2, we will investigate a novel alternative Ca2+ influx pathway that operates in addition to the STIM-ORAI Ca2+ entry pathway in differentiated mouse and human T cells. Aim 1 is broadly relevant to many biological processes and to cancer, since STIM-ORAI proteins mediate store-operated Ca2+ entry not only in immune and haematopoietic cells (T and B lymphocytes, mast cells, platelets) but also in skeletal muscle, smooth muscle, exocrine organs, certain cancers, and almost certainly most other cell types. Aim 2 is designed to establish the molecular identity of the newly documented pathway for Ca2+ entry into immune cells, information that could have major relevance for the treatment of immune disorders and asthma. The two aims dovetail in being directed toward a more complete understanding of physiological Ca2+ signalling in T cells.